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Short Communication |
Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand
Correspondence
Stephen B. Fleming
stephen.fleming{at}stonebow.otago.ac.nz
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ABSTRACT |
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Supplementary figures are available with the online version of this paper.
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MAIN TEXT |
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).
Among its repertoire of immunomodulators, ORFV encodes a homologue of interleukin-10 (IL-10) (Fleming et al., 1997; Haig et al., 2002a). Cellular IL-10 is a multifunctional cytokine that has both immunosuppressive and immunostimulatory properties. The major physiological function of IL-10 is to regulate macrophages activated by pathogens and their products. IL-10 acts in part by reducing the production of proinflammatory mediators from macrophages and suppresses T-cell production of IL-2 and gamma interferon (Moore et al., 2001). In addition, IL-10 inhibits monocyte proliferation (O'Farrell et al., 1998, 2000), but stimulates thymocyte, mast-cell and B-cell proliferation (Moore et al., 2001).
We have reported previously that ORFV IL-10 is functionally similar to cellular IL-10 in that it has the capacity to inhibit cytokine synthesis in ovine and murine monocytes (Fleming et al., 1997; Haig et al., 2002b; Imlach et al., 2002), impairs the maturation of murine and human dendritic cells (Chan et al., 2006; Lateef et al., 2003) and also costimulates mast cells and thymocytes (Fleming et al., 1997; Haig et al., 2002b; Imlach et al., 2002).
The mature polypeptide of ORFV IL-10 is 91 % identical to ovine IL-10, but shows only 75 % identity to human IL-10 (hIL-10) (Fleming et al., 1997). In view of this difference, we were interested to determine whether ORFV IL-10 was likely to be functionally relevant in human ORFV infection and, in particular, whether it was likely to have a role in suppressing inflammation. Here, we investigated the ability of ORFV IL-10 to inhibit cytokine synthesis and proliferation in lipopolysaccharide (LPS)-activated THP-1 monocytes and compared these activities with those of hIL-10.
Firstly, we examined the ability of ORFV IL-10 to inhibit LPS-induced synthesis of proinflammatory cytokines IL-1
and tumour necrosis factor (TNF) in the human monocyte cell line THP-1 (ATCC TIB-202). For protein and transcriptional analyses, THP-1 cells were cultured in RPMI medium containing 0.1 % BSA (1.0x106 cells ml–1). Cells were activated by the addition of LPS derived from Escherichia coli (serotype 055 : B5; Sigma). Recombinant hIL-10 was purchased from R&D Systems. ORFV IL-10 and a mock-purification control were prepared as described previously (Imlach et al., 2002).
For protein analysis, IL-1 and TNF were quantified in cell-cleared supernatants by ELISA using OPT EIA sets (BD Biosciences) following the manufacturer's instructions. ORFV IL-10 significantly inhibited IL-1 production in THP-1 cells treated simultaneously with 10 µg LPS ml–1 and 1–100 ng ORFV IL-10 or hIL-10 ml–1 for 24 h (Fig. 1a). Single-factor analysis of variance was used for this and all subsequent statistical analyses, with significant points of difference, at P < 0.05, determined by using Tukey's test. The greatest inhibitory effects of ORFV IL-10 were seen at concentrations 
10 ng ml–1, where the levels of IL-1 were reduced by 50 % (Fig. 1a). Furthermore, it appeared that equivalent amounts of ORFV IL-10 and hIL-10 had similar inhibitory effects; there was no significant difference (P < 0.05) between IL-10s at any concentration tested (Fig. 1a). In addition, the abilities of ORFV IL-10 and hIL-10 (at 100 ng ml–1) to inhibit the synthesis of TNF, induced with 10 µg LPS ml–1, were similar to those seen for inhibition of IL-1 (P < 0.05) (Fig. 1b). The inhibition of cytokine synthesis was IL-10-specific, as a mock-purification control failed to inhibit TNF production in LPS-activated THP-1 cells (Fig. 1b).
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, 5'-GGCAATGAGGATGACTTGTTCTTT and 5'-CAGAGGTCCAGGTCCTGGAA; TNF, 5'-CTTCTCGAACCCCGAGTGAC and 5'-GAGCTGCCCCTCAGCTTG; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CCCACTCCTCCACCTTT and 5'-TTGCTGTAGCCAAATTCGTTGT, to give amplicons of 77, 68 and 81 bp, respectively. qPCR was carried out with DNA polymerase and SYBR green reagents, and amplifications were performed using an ABI PRISM 7500 sequence detection system. Analyses of qPCR data are described in the manufacturer's manual (PE Applied Biosystems, 1997).
The greatest effects of IL-10 on inhibition of cytokine synthesis at the transcriptional level were seen when THP-1 cells were treated simultaneously with 10 µg LPS ml–1 and 100 ng ORFV IL-10 or hIL-10 ml–1 for 3 h. ORFV IL-10 and hIL-10 inhibited the expression of TNF significantly (by approximately 50 %), whereas IL-1 was inhibited by approximately 30 % (P < 0.05) (Fig. 1c, d
). There was no significant difference between the effects of ORFV IL-10 and hIL-10 (P < 0.05). Again, the inhibition of cytokine synthesis was IL-10-specific, as a mock-purification control failed to inhibit TNF and IL-1 expression in LPS-activated THP-1 cells (Fig. 1c, d).
Curiously, we found that neither ORFV IL-10 nor hIL-10 was able to inhibit the expression or production of TNF and IL-1 when induced by 1 µg LPS ml–1 (Supplementary Fig. S1, available in JGV Online). In view of these findings, we examined the levels of IL-10 receptor 1 (IL-10R1) expression. Previous studies have shown that the upregulation of IL-10R1 mRNA correlates with the cell-surface expression of IL-10R1 and, subsequently, IL-10 responsiveness (Crepaldi et al., 2001; Ding et al., 2001). We therefore examined the levels of IL-10R1 expression at the mRNA level in LPS-treated monocytes by using the primers 5'-TGGGAGAGTTCTGTGTCCAGGT and 5'-AGGCAAAGAAGATGATGACGTTG to give an amplicon of 132 bp. Treatment with 10 µg LPS ml–1 increased the level of IL-10R1 mRNA, whereas lower concentrations of LPS had little or no effect (Supplementary Fig. S2, available in JGV Online). IL-10R1 mRNA levels in THP-1 cells treated with 10 µg LPS ml–1 were significantly higher than background levels at 1 and 3 h post-LPS stimulation (P < 0.05). The increased expression levels of IL-10R1 induced by the higher doses of LPS correlated with the ability of ORFV IL-10 to inhibit the production of TNF and IL-1. This is consistent with the findings of previous studies that proposed the upregulation of IL-10R1 by LPS to be a priming mechanism that allows cells to respond rapidly to IL-10 (Cassatella et al., 2005; Murray, 2006).
We then examined the ability of ORFV IL-10 to inhibit monocyte proliferation. THP-1 cells, cultured in RPMI medium containing 5 % fetal calf serum (2.5x105 cells ml–1), were treated for 24 h with or without 10 µg LPS ml–1, then incubated with various concentrations of ORFV IL-10 or hIL-10 for 48 h. DNA synthesis was quantified by the addition of 1 µCi (37 kBq) [3H]thymidine during the final 4 h incubation, followed by harvesting using an automated cell harvester (Tomtec). Incorporated [3H]thymidine was measured by -counting (Top Count NXT scintillation counter; Canberra Packard).
We found that neither hIL-10 nor ORFV IL-10 had an effect on the proliferation of THP-1 cells at any concentration (Supplementary Fig. S3, available in JGV Online). In the presence of LPS, however, we found that hIL-10 inhibited proliferation significantly, by approximately 30 % (P < 0.05), from 10 ng ml–1 (Fig. 2). This suggests that inhibition of monocyte proliferation by IL-10 is also dependent on the level of IL-10R1. Unlike hIL-10, ORFV IL-10 had no effect on THP-1 proliferation at 10 ng ml–1, but did inhibit monocyte proliferation significantly, by 28 % (P < 0.05), at the higher concentration of 100 ng ml–1 (Fig. 2). The effects of ORFV IL-10 on monocyte proliferation were significantly different (P < 0.05) from those of hIL-10 from 1 to 100 ng ml–1 (Fig. 2). This is in contrast to the equivalent abilities of ORFV IL-10 and hIL-10 to inhibit LPS-induced cytokine synthesis at these doses.
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; Zdanov et al., 1996) by using SWISS-MODEL and the SwissPdbViewer protein-modelling program (version 3.7; Guex & Peitsch, 1997). The structure of ORFV IL-10 was very similar to that of hIL-10, conserving the six 
-helical segments and the six core cysteine residues (Fig. 3a, b).
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; Spencer et al., 1998), and signal transduction can only occur in cells expressing both subunits (Spencer et al., 1998). The ternary IL-10 signalling complex is assembled in a sequential order with the IL-10–IL-10R1 interaction occurring first, followed by the engagement of the IL-10R2 chain. The high-affinity interaction of IL-10 with IL-10R1 produces a conformational change that allows the low-affinity binding of IL-10R2, completing the signalling complex, which then activates the JAK/STAT signalling pathway (Kotenko et al., 1997; Usacheva et al., 2002; Yoon et al., 2006).
Analysis of the crystal structure of the hIL-10–IL-10R1 complex has shown that the IL-10 dimer binds symmetrically to two soluble IL-10R1 chains (Josephson et al., 2001). The structure of the hIL-10–IL-10R1 complex interacting with two IL-10R2 chains has also been predicted (Pletnev et al., 2005; Yoon et al., 2006). Amino acids located in helix A, the A–B loop and helix F of hIL-10 contact IL-10R1, and amino acids located in the N terminus, helix A, the C–D loop and helix D of hIL-10 contact IL-10R2 (Fig. 3a-d). Comparison of ORFV IL-10 with hIL-10 reveals that nine of the 11 amino acids of hIL-10 that are thought to contact IL-10R1 are conserved in ORFV IL-10, whilst only six of the 11 amino acids thought to contact IL-10R2 are identical to those in hIL-10 (Fig. 3a, c).
Cellular IL-10 inhibits macrophage proliferation by STAT3-dependent signalling mechanisms (O'Farrell et al., 1998, 2000). Recently, it has been shown that STAT3 activation is dependent on three amino acid residues (Asn39, Met40 and Arg50) of cellular IL-10 that are critical in interactions with IL-10R2 (Yoon et al., 2006). The lack of two of the three critical residues in ORFV IL-10 (Asn39 and Arg50) (Fig. 3a, c) may explain our observation that ORFV IL-10 shows a markedly reduced ability to inhibit monocyte proliferation compared with hIL-10.
Studies linking specific signalling events with anti-inflammatory processes are very limited and contradictory, but it appears that cellular IL-10 can inhibit macrophage activation and proliferation by distinct STAT3-dependent and -independent pathways (O'Farrell et al., 1998, 2000). Two recent studies have demonstrated that the ability of cellular IL-10 to disrupt the stability of LPS-induced mRNA is, in part, mediated through a STAT3-independent/SOCS3-dependent pathway (Qasimi et al., 2006; Williams et al., 2004). Our data also suggest that the ability of ORFV IL-10 to inhibit THP-1 monocyte activation may not be dependent on IL-10R2–ORFV IL-10 binding. Further studies are in progress to examine the role of the IL-10 receptors and the SOCS3- or STAT3-dependent pathways in ORFV IL-10-induced monocyte activation.
Our study has revealed that ORFV IL-10 may have a reduced capacity to modulate monocyte growth in the human host, although its effects on limiting the expression of immune effector molecules are equally as potent as those of cellular IL-10. Clearly, ORFV IL-10 has adapted to its ovine host and we predict from its close similarity to ovine IL-10 (Fleming et al., 1997) that ORFV IL-10 will probably have the capacity to inhibit ovine monocyte proliferation. It is clear that, in some cases, ORFV-encoded virulence factors are either not active in humans, as is the case with the ORFV granulocyte–macrophage colony-stimulating factor/IL-2-binding protein (Deane et al., 2000), or, as in the case of ORFV IL-10, lack at least one activity of their cellular counterparts. This highlights the problems that large, complex poxviruses have when adapting to a new host species, in that their immunomodulators are tailored to operate in their natural host and may have no or partial activity in other hosts that the virus infects.
In conclusion, our results show that ORFV IL-10 has the ability to inhibit cytokine synthesis in THP-1 monocytes activated by LPS and that this activity is equivalent to that of hIL-10. In contrast, ORFV IL-10 displays a reduced ability to inhibit the proliferation of THP-1 monocytes compared with hIL-10, and we have identified structural features of ORFV IL-10 that may explain this observation. It appears that ORFV IL-10 is likely to be a virulence factor in human ORFV infection, but lacks at least one of the activities of hIL-10 that is involved in limiting inflammation. Our results also highlight the utility of viral molecules as tools to study the principles of cell biology and immunology.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chan, A., Baird, M., Mercer, A. A. & Fleming, S. B. (2006). Maturation and function of human dendritic cells are inhibited by orf virus-encoded interleukin-10. J Gen Virol 87, 3177–3181.
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Received 6 December 2006;
accepted 5 February 2007.
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) compared with the LPS-only control is indicated (P<0.05).
